Tuberculosis (TB) is the second leading cause of death among communicable diseases worldwide. The current front-line TB drug isoniazid acts by inhibiting the biosynthesis of an Mtb outer membrane lipid, mycolic acid, underscoring the importance of the outer membrane in virulence and survival. However, widespread resistance to isoniazid and other front-line therapies requires the development of new drugs to combat multidrug- and extensively drug-resistant organisms (MDR/XDR-TB). Novel therapies that inhibit multiple targets by combining two or more agents are highly desirable to increase the rapidity and efficacy of treatment and slow the emergence of drug resistance. Therefore, there is an urgent need to identify the effects of repressing two genes simultaneously. The long-term goals are to elucidate (1) the mechanisms of outer membrane biogenesis and (2) the synergism between these pathways in promoting Mtb virulence and survival. The objective of this application is to facilitate these goals through the development of novel gene regulation systems for mycobacteria. The overall strategy is to optimize riboswitches for efficient, inducible gene expression and repression. The rationale for the proposed research is that inducible gene regulation tools based on riboswitches will enable, for the first time, the independent and simultaneous experimental control of two genes both in vitro and in vivo. Thus, the following specific aims are proposed: (1) To optimize inducible riboswitches to turn gene expression on or off; (2) to demonstrate riboswitch regulation of endogenous mycobacterial genes; and (3) to determine an appropriate dosing regimen for the effector molecule in mice. Riboswitches that respond to different effector molecules have been validated in in vitro culture and macrophage infection models in the applicant's laboratory. The approach is to identify optimal riboswitches by screening libraries of randomized sequence variants. The research proposed in this application is innovative because it departs from the status quo via the application of riboswitches to mycobacterial gene regulation and will overcome a current technical hurdle in the systematic evaluation of dual knockout phenotypes. This contribution is significant because the elucidation of dual knockout phenotypes, whether synthetic lethal or suppressive, will help create maps of metabolic and signaling pathways and their interactions, and also facilitate the functional assignment of the hundreds of Mtb genes of unknown function.